WS7622A, B, C and D substances, derivatives thereof, processes for preparation thereof and use for treatment of pulmonary emphysema and adult respiratory distress syndrome

ABSTRACT

A method of treating pulmonary emphysema or adult respiratory distress syndrome in a subject in need thereof which comprises administering to the subject an effective amount of WS7622A, B, C and/or D, derivatives thereof, or their pharmaceutically acceptable salt.

This is a continuation of application Ser. No. 07/482,822, filed on Feb.21, 1990 now U.S. Pat. No. 5,021,240.

This invention relates to new WS7622A, B, C and D substances andderivatives thereof.

More particularly, this invention relates to new WS7622A, B, C and Dsubstances, derivatives thereof and their pharmaceutically acceptablesalts which have an human leukocyte elastase-inhibiting activity, toprocesses for preparation thereof, and to a pharmaceutical compositioncomprising the same and to a method of use thereof.

The WS7622A, B, C and D substances can be produced by culturing aWS7622A, B, C and D substances-producing strain of the genusStreptomyces in a nutrient medium.

THE MICROORGANISM

The microorganism which can be used for the production of WS7622A, B, Cand D substances is a WS7622A, B, C and D substances-producing strainbelonging to the genus Streptomyces, among which Streptomycesresistomycificus No. 7622 has been newly isolated from a soil samplecollected at Hirono-cho, Fukushima-ken, Japan.

A lyophilized sample of the newly isolated Streptomyces resistomycificusNo. 7622 was firstly deposited with International Depositary Authorityunder the Budapest Treaty, the Fermentation Research Institute, Agencyof Industrial Science and Technology (1-3, Higashi 1 chome, Tsukuba-shi,Ibaraki-ken, 305 Japan) under the accession number of FERM P-2306 (thedeposited date: Feb. 23, 1989), as Streptomyces sp. No. 7622, and thenthis strain was named as Streptomyces resistomycificus No. 7622.

It is to be understood that the production of the novel WS7622A, B, Cand D substances is not limited to the use of the particular organismdescribed herein, which is given for the illustrative purpose only. Thisinvention also includes the use of any mutants which are capable ofproducing WS7622A, B, C and D substances including natural mutants aswell as artificial mutants which can be produced from the describedorganism by conventional means such as irradiation of X-ray,ultra-violet radiation, treatment withN-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine, and the like.

The Streptomyces resistomycificus No. 7622 has the followingmorphological, cultural, biological and physiological characteristics.

(1) Morphological Characteristics:

The methods described by Shirling and Gottlieb ¹) were employed for thistaxonomic study.

Morphological observations were made with light and electron microscopeson cultures grown at 30° C. for 14 days on oatmeal agar, yeastextract-malt extract agar and inorganic salts-starch agar.

The vegetative mycelium developed well without fragmentation. The aerialmycelium branched monopodially and formed spiral chains andrectus-flexibilis chains of spores with more than 30 spores per chain.The spores had a smooth surface and were oval in shape with a size of0.7-0.9×0.8-1.1 μm. Sclerotic granules, sporangia and zoospores were notobserved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an infrared absorption spectrum of WS7622A.

FIG. 2 is an NMR spectrum of WS7622A.

FIG. 3 is a ¹³ C NMR spectrum of WS7622A.

FIG. 4 is an infrared absorption spectrum of WS7622B.

FIG. 5 is an NMR spectrum of WS7622B.

FIG. 6 is a ¹³ C NMR spectrum of WS7622B.

FIG. 7 is an infrared absorption spectrum of WS7622C.

FIG. 8 is an NMR spectrum of WS7622C.

FIG. 9 is a ¹³ C NMR spectrum of WS7622C.

FIG. 10 is an infrared absorption spectrum of WS7622D.

FIG. 11 is an NMR spectrum of WS7622D.

FIG. 12 is a ¹³ C NMR spectrum of WS7622D.

(2) Cultural Characteristics:

Cultural characteristics were observed on ten kinds of media describedby Shirling and Gottlieb as mentioned above, and by Waksman²).

The incubation was carried out at 30° C. for 21 days. The color namesused in this study were taken from Methuen Handbook of Colour³). Theresults are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Cultural characteristics of the strain No. 7622                               Medium         Cultural characteristics                                       ______________________________________                                        Yeast extract-malt                                                                           G: good                                                        extract agar   A: moderate, bluish gray(22E2)                                                R: brown(6F4)                                                                 S: none                                                        oatmeal agar   G: good                                                                       A: moderate, bluish gray(22D2)                                                R: brown(6F6)                                                                 S: none                                                        inorganic salts-                                                                             G: good                                                        starch agar    A: abundant, brownish gray(7E2)                                               R: violet brown(11F6)                                                         S: none                                                        glycerin-asparagine                                                                          G: good                                                        agar           A: abundant, brownish gray(6E2)                                               R: grayish orange(5B5)                                                        S: none                                                        peptone-yeast  G: moderate                                                    extract-iron agar                                                                            A: none                                                                       R: brown(6F6)                                                                 S: dark brown                                                  tyrosine agar  G: good                                                                       A: poor, turquoise gray(24E2)                                                 R: dark brown(7F8)                                                            S: none                                                        glucose-asparagine                                                                           G: moderate                                                    agar           A: none                                                                       R: yellowish white(4A2)                                                       S: none                                                        nutrient agar  G: poor                                                                       A: none                                                                       R: yellowish white(4A2)                                                       S: none                                                        Bennet agar    G: good                                                                       A: poor, bluish gray(22E3)                                                    R: brownish orange(5C5)                                                       S: none                                                        sucrose-nitrate                                                                              G: good                                                        agar           A: none                                                                       R: grayish Magenta(13D4)                                                      S: none                                                        ______________________________________                                         Abbreviation:                                                                 G = growth, A = aerial mycelium, R = reverse side color, S = soluble          pigment                                                                  

The aerial mycelium was bluish gray to brownish gray. Reverse side ofgrowth was brown on yeast extract-malt extract agar and oatmeal agar,violet brown on inorganic salts-starch agar, and grayish Magenta onsucrose-nitrate agar. Melanoid pigments were produced, but other solublepigments were not produced.

(3) Cell wall type:

The cell wall analysis was performed by the methods of Becker et al⁴)and Yamaguchi⁵).

Analysis of whole cell hydrolysates of strain No. 7622 showed thepresence of LL-diaminopimelic acid. Accordingly, the cell wall of thisstrain is classified as type I.

(4) Biological and physiological properties:

Physiological properties and utilization of carbon sources are shown inTables 2 and 3, respectively.

Temperature range for growth was determined on yeast-malt extract agarusing a temperature gradient incubator TN-3 (made by Advantec Toyo Co.,Ltd.).

Utilization of carbon sources was examined according to the method ofPridham and Gottlieb⁶).

                  TABLE 2                                                         ______________________________________                                        Physiological properties of strain No. 7622                                   Conditions             Characteristics                                        ______________________________________                                        temperature range for growth                                                                         12° C.-34° C.                            optimum temperature for growth                                                                       27° C.                                          gelatin liquefaction   positive                                               milk coagulation       negative                                               milk peptonization     positive                                               starch hydrolysis      positive                                               production of melanoid pigments                                                                      positive                                               decomposition of cellulose                                                                           negative                                               NaCl tolerance         >3% and <4%                                            ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Carbon utilization of the strain No. 7622                                            Compounds                                                                             Growth                                                         ______________________________________                                               D-glucose                                                                             +                                                                     sucrose +                                                                     D-xylose                                                                              ±                                                                  D-fructose                                                                            +                                                                     L-rhamnose                                                                            +                                                                     raffinose                                                                             -                                                                     L-arabinose                                                                           +                                                                     inositol                                                                              +                                                                     mannitol                                                                              +                                                              ______________________________________                                         +: utilization                                                                ±: doubtful utilization                                                    -: no utilization                                                        

Based on the morphological and physiological characteristics describedabove, strain No. 7622 is considered to belong to the genusStreptomyces.⁷)

So, strain N. 7622 was compared with Streptomyces species described inliterature.⁸⁻¹²) As a result, it was found that the strain proved toclosely resemble Streptomyces resistomycificus IF012814 in detail.There, it was found that the properties of both strains were almostidentical except a few differences.

Table 4 shows the differences between the two strains.

                  TABLE 4                                                         ______________________________________                                        Differences between strain No. 7622 and                                       Streptomyces resistomycificus IF012814                                                       Characteristics                                                Conditions       IF012814    No. 7622                                         ______________________________________                                        growth on sucrose-nitrate agar                                                                 poor        good                                             utilization of sucrose                                                                         no utilization                                                                            utilization                                      temperature range for growth                                                                   14-37° C.                                                                          12-34° C.                                 NaCl tolerance   >4% and <6% >3% and <4%                                      ______________________________________                                    

It is considered to be proper that these differences are too small toregard strain No. 7622 as a different species.

Therefore, we identified the strain as Streptomyces resistomycificus anddesignated it Streptomyces resistomycificus No. 7622.

1) Shirling, E. B. and D. Gottlieb: Methods for characterization ofStreptomyces species. International Journal of Systematic Bacteriology,16, 313-340, 1966

2) Waksman, S. A.: The actinomycetes Vol. 2: Classification,identification and description of genera and species : The Williams andWilkins Co., Baltimore, 1961

3) Kornerup, A. and J. H. Wanscher: Methuen Handbook of Colour, Methuen,London, 1978

4) Becker, B., M. P. Lechevalier, R. E. Gordon and H. A. Lechevalier:Rapid differentiation between Nocardia and Streptomyces by paperchromatography of whole-cell hydrolysates: Appl. Microbiol. 12, 421-423,1964

5) Yamaguchi, T.: Comparison of the cell wall composition ofmorphologically distinct actinomycetes: J. Bacteriol. 89, 444-453, 1965

6) Pridham, T. G. and D. Gottlieb: The utilization of carbon compoundsby some Actinomycetales as an aid for species determination: J.Bacteriol. 56: 107-114, 1948

7) Buchanan, R. E. and N. E. Gibbons: Bergey's Manual of DeterminativeBacteriology, 8th edition: The Williams and Wilkins Co., Baltimore, 1974

8) Shirling, E. B. and D. Gottlieb: Cooperative description of typeculture of Streptomyces. 2. Species descriptions from first study.Intern. J. Syst. Bacteriol. 18: 69-189, 1968

9) Shirling, E. B. and D. Gottlieb: Cooperative description of typeculture of Streptomyces. 3. Additional species descriptions from firstand second studies. Intern. J. Syst. Bacteriol. 18: 279-392, 1968

10) Shirling, E. B. and D. Gottlieb: Cooperative description of typeculture of Streptomyces. 4. Species descriptions from the second, thirdand forth studies. Intern. J. Syst. Bacteriol. 19: 391-512, 1969

11) Skerman, V. B. D.; V. McGowan & P. H. A. Sneath: Approved list ofbacterial names. Intern. J. Syst. Bacteriol. 30: 225-420, 1980

12) Moore, W. E. C., E. P. Cato & L. V. H. Moore: Index of Bacterial andYeast Nomenclatural Changes Published in the I. J. S. B. Since the 1980Approved Lists of Bacterial Names. Intern. J. Syst. Bacteriol. 35:382-407, 1985

WS7622A, B, C and D SUBSTANCES

The WS7622A, B, C and D substances are produced when WS7622A, B, C and Dsubstances-producing strain belonging to the genus Streptomyces is grownin a nutrient medium containing sources of assimilable carbon andnitrogen under aerobic conditions (e.g. shaking culture, submergedculture, etc.). The medium may be either synthetic, semi-synthetic ornatural.

Preferred carbon sources may be glucose, mannose, glycerin, molasses,starch, starch hydrolysate and so on, and preferred nitrogen sources maybe meat extract, casein hydrolysate, peptone, gluten meal, corn meal,cottonseed meal, soybean meal, corn steep liquor, dried yeast, ammoniumphosphate, ammonium sulfate, urea and so on. There may also beincorporated inorganic salts such as the phosphates, chlorides and othersalts of metals, e.g. disodium hydrogen phosphate, potassium dihydrogenphosphate, calcium carbonate, ferrous sulfate magnesium sulfate, coppersulfate, zinc sulfate, manganese chloride, magnesium chloride, etc. Ifcopious foaming is encountered during fermentation, a deforming agentsuch as vegetable oils, e.g. soybean oil, linseed oil, etc., higheralcohols, e.g. octadecanol, may be added in suitable amounts.

The fermentation is preferably conducted at around 30° C. for 50 to 200hours.

From the above-mentioned fermentation conditions, the optimum set ofconditions is selected according to the characteristics of the strain ofmicroorganism employed.

Since a major portion of the WS7622A, B, C and D substances thusproduced in the culture broth is present intracellularly, the culturedbroth is first filtered. To the mycelial cake obtained there is added asuitable solvent such as acetone and the desired compound is thenseparated and purified from the resultant mixture by the procedureemployed commonly in the production of antibiotics in general. Forexample, there may be employed such procedures as concentration underreduced pressure, freeze drying, solvent extraction, pH adjustment,treatment with an anion exchange resin, cation exchange resin, nonionicadsorbent resin, etc., treatment with an adsorbent agent such asactivated carbon, silicic acid, silica gel or alumina, crystallization,and recrystallization, either singly or in an optional combination.

The WS7622 components, A, B, C and D can be separated from the culturedbroth, for example, by the following High Performance LiquidChromatography data:

Column: Yamamura Chemical Institute, Kyoto, A-302 (ODS 6φ×150 mm)

Mobile phase: CH₃ OH--CH₃ CN--0.3% aq.H₃ PO₄ (10:1:10)

Detection: 210 nm

Flow rate: 1 ml/min

Retention Time (min):

WS7622A: 6.93

WS7622B: 10.08

WS7622C: 5.30

WS7622D: 4.38

The WS7622A, B, C and D substances produced in the culture broth can beisolated in its free form or if desired, in the form of apharmaceutically acceptable salt, respectively. For isolating thesesubstances in the form of a pharmaceutically acceptable salt, thedesired compound obtained from the mycelial extract is treated with abase such as an inorganic base, e.g. an alkali metal compound (e.g.sodium hydroxide, potassium hydroxide, etc.), an alkaline earth metalcompound (e.g. calcium hydroxide, magnesium hydroxide, etc.), aninorganic base, e.g. ammonia, etc., an organic base (e.g. triethylamine,dicyclohexylamine, etc.) or an acid such as an inorganic acid (e.g.hydrochloric acid, sulfuric acid, phosphoric acid, etc.) or an organicacid (e.g. formic acid, acetic acid, p-toluenesulfonic acid, citricacid, oxalic acid, etc.), whereby each of the corresponding salt ofWS7622A, B, C and D substances can be obtained.

Each of the salt of WS7622A, B, C and D substances thus obtained can bereconverted to free WS7622A, B, C and D substances in the per seconventional manner.

(1) WS7622A SUBSTANCE

The WS7622A substance is a new substance and has the followingphysico-chemical properties.

Physico-chemical properties of WS7622A substance:

Appearance: Colorless prism

Nature of substance: acidic

Color reaction:

positive; cerium sulfate, iodine vapor

negative; ninhydrin, Molish

Solubility:

soluble; methanol, ethanol, n-butanol

sparingly soluble; chloroform, acetone, ethyl acetate

insoluble; water, n-hexane

Thin Layer Chromatography (TLC):

chloroform-methanol (5:1, v/v): Rf 0.51

acetone-methanol (10:1): 0.62

(Kiesel gel 60 F254 silica gel plate, Merck)

Melting point: 250°-252° C. (dec.)

Specific rotation: [α]_(D) ²³ +36° (c=1.0, MeOH)

    ______________________________________                                        UV spectrum: γ.sub.max.sup.MeOH                                                                   287 nm (ε = 3600)                                        γ.sub.max.sup.MeOH--HCl                                                              287 nm                                                           γ.sub.max.sup.MeOH--NaOH                                                             298 nm                                              ______________________________________                                    

Molecular formula: C₄₇ H₆₃ N₉ O₁₃

Elemental analysis: Calcd. (C₄₇ H₆₃ N₉ O₁₃ ·2H₂ O); C 56.56, H 6.77, N12.63%. Found; C 56.65, H 6.62, N 12.27%.

Molecular weight: FAB-MS m/z 984 (M+Na)⁺

Infrared absorption spectrum (attached FIG. 1): ν_(max) ^(KBr) 3400,3300, 3060, 2980, 2940, 1735, 1710, 1690, 1670, 1660, 1640, 1540, 1520,1470, 1380, 1330, 1300, 1260, 1220, 1200, 1160, 1130, 1090, 1000, 980,940, 920 cm⁻¹

    ______________________________________                                        .sup.1 H Nuclear magnetic resonance spectrum (attached FIG. 2):               (400 MHz, CD.sub.3 OD) δ                                                ______________________________________                                        7.22-7.09         (3H, m)                                                     6.88-6.77         (3H, m)                                                     6.74              (1H, s)                                                     6.46              (1H, s)                                                     5.46              (1H, m)                                                     5.18              (1H, s)                                                     4.85              (1H, s)                                                     4.77              (1H, m)                                                     4.65              (1H, m)                                                     4.50              (1H, m)                                                     3.96              (1H, m)                                                     3.91              (1H, d, J=9Hz)                                              3.60-3.47         (2H, m)                                                     3.03              (1H, m)                                                     2.90              (3H, s)                                                     2.86              (1H, m)                                                     2.59-2.49         (2H, m)                                                     2.39              (1H, m)                                                     2.29-2.16         (2H, m)                                                     2.00              (1H, m)                                                     1.84              (1H, m)                                                     1.74              (3H, d, J=6Hz)                                              1.72-1.53         (4H, m)                                                     1.44              (3H, d, J=6Hz)                                              1.12              (1H, m)                                                     1.10              (6H, d, J=6Hz)                                              0.99              (3H, d, J=6Hz)                                              0.94              (3H, d, J=6Hz).                                             ______________________________________                                    

    ______________________________________                                        .sup.13 C Nuclear magnetic resonance spectrum (attached FIG. 3):              (100 MHz, CD.sub.3 OD) δ                                                ______________________________________                                        179.7                (s)                                                      176.3                (s)                                                      174.7                (s)                                                      173.3                (s)                                                      172.4                (s)                                                      171.4                (s)                                                      170.3                (s)                                                      165.8                (s)                                                      160.2                (s)                                                      145.7                (s)                                                      145.6                (s)                                                      137.5                (s)                                                      134.0                (d)                                                      131.4                (s)                                                      130.6                (d) × 2                                            129.8                (s)                                                      129.1                (d) × 2                                            129.1                (s)                                                      127.6                (d)                                                      119.1                (d)                                                      118.0                (d)                                                      76.0                 (d)                                                      73.4                 (d)                                                      63.1                 (d)                                                      61.4                 (d)                                                      57.1                 (d)                                                      53.6                 (d)                                                      52.7                 (d)                                                      50.5                 (d)                                                      39.9                 (t)                                                      36.1                 (t)                                                      35.8                 (d)                                                      31.8                 (q)                                                      31.0                 (t)                                                      30.8                 (d)                                                      29.9                 (t)                                                      29.7                 (t)                                                      25.2                 (t)                                                      22.3                 (t)                                                      20.2                 (q)                                                      20.0                 (q) × 2                                            19.7                 (q)                                                      19.5                 (q)                                                      13.3                 (q)                                                      ______________________________________                                    

(2) WS7622B SUBSTANCE

The WS7622B substance is a new substance and has the followingphysico-chemical properties.

Physico-chemical properties of WS7622B substance:

Appearance: Colorless needles

Nature of substance: acidic

Color reaction:

positive; Cerium sulfate, iodine vapor, ferric chloride

negative; ninhydrin, Molish, Dragendorff

Solubility:

soluble; methanol, ethanol, n-butanol

sparingly soluble; chloroform, acetone

insoluble; water, n-hexane

Thin Layer Chromatography (TLC):

chloroform-methanol (5:1, V/V): Rf 0.55

(Kiesel gel 60 F254 silica gel plate, Merck)

Melting point: 248°-250° C. (dec.)

Specific rotation: [α]_(D) ²³ +39° (C=1.0, MeOH)

    ______________________________________                                        UV Spectrum: γ.sub.max.sup.MeOH                                                                   287 nm (ε = 3800)                                        γ.sub.max.sup.MeOH--HCl                                                              287 nm                                                           γ.sub.max.sup.MeOH--NaOH                                                             299 nm                                              ______________________________________                                    

Molecular formula: C₄₈ H₆₅ N₉ O₁₃

Elemental analysis: Calcd. (C₄₈ H₆₅ N₉ O₁₃.3H₂ O); C 55.96, H 6.95, N12.24%. Found; C 55.84, H 7.05, N 12.12%.

Molecular weight: FAB-MS m/z 998 (M+Na)⁺

Infrared absorption spectrum (attached FIG. 4): ν_(max) ^(KBr) 3400,3300, 2960, 1735, 1680, 1660, 1640, 1540, 1520, 1460, 1400, 1380, 1330,1290, 1250, 1200, 1180, 1150, 1130, 1080, 1050, 1000, 980, 940, 920 cm⁻¹

    ______________________________________                                        .sup.1 H Nuclear magnetic resonance spectrum (attached FIG. 5):               (400 MHz, DMSO-d.sub.6) δ                                               ______________________________________                                        9.48            (1H, broad s)                                                 9.03            (1H, broad s)                                                 8.76            (1H, broad s)                                                 8.30            (1H, broad d, J=6Hz)                                          7.77            (1H, d, J=7Hz)                                                7.21            (1H, d, J=8Hz)                                                7.20-7.11       (3H, m)                                                       6.97            (1H, broad d, J=7Hz)                                          6.80            (2H, d, J=8Hz)                                                6.72            (1H, broad s)                                                 6.67            (1H, s)                                                       6.63            (1H, q, J=7Hz)                                                6.37            (1H, s)                                                       5.48            (1H, m)                                                       5.40            (1H, m)                                                       5.09            (1H, m)                                                       4.77            (1H, m)                                                       4.64            (1H, m)                                                       4.38            (1H, m)                                                       4.31            (1H, m)                                                       3.87-3.80       (2H, m)                                                       3.40-3.30       (2H, m)                                                       2.95            (1H, m)                                                       2.79            (3H, s)                                                       2.65            (1H, m)                                                       2.40-2.20       (4H, m)                                                       2.00            (1H, m)                                                       1.87            (1H, m)                                                       1.73            (1H, m)                                                       1.65            (3H, d, J=7Hz)                                                1.65-1.40       (5H, m)                                                       1.32            (3H, d, J=6Hz)                                                1.27            (1H, m)                                                       0.97            (3H, d, J=6Hz)                                                0.97            (1H, m)                                                       0.91            (3H, d, J=6Hz)                                                0.88            (3H, d, J=6Hz)                                                0.81            (3H, t, J=7Hz)                                                ______________________________________                                    

    ______________________________________                                        .sup.13 C Nuclear magnetic resonance spectrum (attached FIG. 6):              (100 MHz, DMSO-d.sub.6) δ                                               ______________________________________                                        175.6                (s)                                                      174.8                (s)                                                      172.0                (s)                                                      170.9                (s)                                                      170.6                (s)                                                      170.3                (s)                                                      168.7                (s)                                                      162.5                (s)                                                      157.4                (s)                                                      144.2                (s)                                                      144.2                (s)                                                      136.5                (s)                                                      131.1                (d)                                                      130.7                (s)                                                      129.6                (d) × 2                                            128.4                (s)                                                      127.9                (d) × 2                                            127.8                (s)                                                      126.4                (d)                                                      118.9                (d)                                                      116.8                (d)                                                      74.2                 (d)                                                      72.1                 (d)                                                      61.0                 (d)                                                      59.8                 (d)                                                      55.2                 (d)                                                      51.6                 (d)                                                      50.8                 (d)                                                      48.6                 (d)                                                      40.9                 (d)                                                      38.2                 (t)                                                      35.0                 (t)                                                      30.8                 (q)                                                      29.8                 (t)                                                      28.8                 (d)                                                      28.4                 (t)                                                      28.1                 (t)                                                      27.1                 (t)                                                      23.3                 (t)                                                      21.2                 (t)                                                      19.6                 (q)                                                      19.1                 (q)                                                      19.0                 (q)                                                      17.7                 (q)                                                      12.9                 (q)                                                      11.9                 (q)                                                      ______________________________________                                    

(3) WS7622C SUBSTANCE

The WS7622C substance is a new substance and has the followingphysico-chemical properties.

Physico-chemical properties of WS7622C substance:

Appearance: Colorless needles

Nature of substance: acidic

Color reaction:

positive; Cerium sulfate, iodine vapor, ferric chloride

negative; ninhydrin, Molish, Dragendorff

Solubility:

soluble; methanol, ethanol

sparingly soluble; chloroform, acetone, ethyl acetate

insoluble; water, n-hexane

Thin Layer Chromatography (TLC):

chloroform-methanol (4:1, V/V): Rf 0.56

(Kiesel gel 60 F254 silica gel plate, Merck)

Melting point: 250°-252° C. (dec.)

Specific rotation: [α]_(D) ²³ +36° (C=0.5, MeOH)

    ______________________________________                                        UV spectrum: γ.sub.max.sup.MeOH                                                                   287 nm (ε = 3500)                                        γ.sub.max.sup.MeOH--HCl                                                              287 nm                                                           γ.sub.max.sup.MeOH--NaOH                                                             298 nm                                              ______________________________________                                    

Molecular formula: C₄₆ H₆₁ N₉ O₁₃

Elemental analysis: Calcd. (C₄₆ H₆₁ N₉ O₁₃.6H₂ O); C 52.31, H 6.97, N11.94%. Found; C 51.95, H 6.66, N 11.77%.

Molecular weight: FAB-MS m/z 970 (M+Na)⁺

Infrared absorption spectrum (attached FIG. 7): ν_(max) ^(KBr) 3400,3300, 2980, 1740, 1700, 1660, 1640, 1540, 1470, 1450, 1410, 1335, 1260,1230, 1200, 1160, 1140, 1070, 1010, 980, 940, 920, 880 cm⁻¹

    ______________________________________                                        .sup.1 H Nuclear magnetic resonance spectrum (attached FIG. 8):               (400 MHz, CD.sub.3 OD) δ                                                ______________________________________                                        7.21-7.10       (3H, m)                                                       6.86-6.77       (3H, m)                                                       6.75            (1H, s)                                                       6.47            (1H, s)                                                       5.46            (1H, m)                                                       5.18            (1H, m)                                                       4.85            (1H, s)                                                       4.74            (1H, m)                                                       4.65            (1H, m)                                                       4.51            (1H, m)                                                       3.94            (1H, m)                                                       3.90            (1H, d, J=10Hz)                                               3.58-3.46       (2H, m)                                                       3.02            (1H, m)                                                       2.90            (3H, s)                                                       2.86            (1H, m)                                                       2.55            (1H, m)                                                       2.38            (1H, dd, J=14 and 11Hz)                                       2.28            (2H, q, J=7Hz)                                                2.30-2.16       (2H, m)                                                       1.99            (1H, m)                                                       1.84            (1H, m)                                                       1.75            (3H, d, J=7Hz)                                                1.78-1.54       (4H, m)                                                       1.44            (3H, d, J=6.5Hz)                                              1.13            (3H, t, J=7Hz)                                                1.12            (1H, m)                                                       1.01            (3H, d, J=6.5Hz)                                              0.97            (3H, d, J=6.5Hz)                                              ______________________________________                                    

    ______________________________________                                        .sup.13 C Nuclear magnetic resonance spectrum (attached FIG. 9):              (100 MHz, CD.sub.3 OD) δ                                                ______________________________________                                        176.7                (s)                                                      176.4                (s)                                                      174.7                (s)                                                      173.4                (s)                                                      172.5                (s)                                                      171.4                (s)                                                      170.4                (s)                                                      165.8                (s)                                                      160.4                (s)                                                      145.8                (s)                                                      145.7                (s)                                                      137.5                (s)                                                      134.0                (d)                                                      131.4                (s)                                                      130.6                (d) × 2                                            129.8                (s)                                                      129.1                (d) × 2                                            129.1                (s)                                                      127.7                (d)                                                      119.1                (d)                                                      118.0                (d)                                                      76.0                 (d)                                                      73.5                 (d)                                                      63.1                 (d)                                                      61.4                 (d)                                                      57.1                 (d)                                                      53.7                 (d)                                                      52.7                 (d)                                                      50.5                 (d)                                                      39.9                 (t)                                                      36.1                 (t)                                                      31.8                 (q)                                                      31.0                 (t)                                                      30.8                 (d)                                                      29.9                 (t)                                                      29.7                 (t)                                                      29.7                 (t)                                                      25.3                 (t)                                                      22.3                 (t)                                                      20.2                 (q)                                                      19.4                 (q)                                                      19.4                 (q)                                                      13.3                 (q)                                                      10.3                 (q)                                                      ______________________________________                                    

(4) WS7622D SUBSTANCE

The WS7622D substance is a new substance and has the followingphysico-chemical properties.

Physico-chemical properties of WS7622D substance:

Appearance: Colorless needles

Nature of substance: acidic

Color reaction:

positive; Cerium sulfate, iodide vapor, ferric chloride

negative; ninhydrin, Molish, Dragendorff

Solubility:

Soluble; methanol, ethanol

sparingly soluble; water, chloroform

insoluble; n-hexane

Thin Layer Chromatography (TLC):

chloroform-methanol (4:1, V/V): Rf 0.45

(Kiesel gel 60 F254 silica gel plate, Merck)

Melting point: 250°-252° C. (dec.)

Specific rotation: [α]_(D) ²⁴ +35.8° (C=0.5, MeOH)

    ______________________________________                                        UV spectrum: γ.sub.max.sup.MeOH                                                                   287 nm (ε = 3640)                                        γ.sub.max.sup.MeOH--HCl                                                              287 nm                                                           γ.sub.max.sup.MeOH--NaOH                                                             298 nm                                              ______________________________________                                    

Molecular formula: C₄₅ H₅₉ N₉ O₁₃

Elemental analysis: Calcd. (C₄₅ H₅₉ N₉ O₁₃.6H₂ O); C 51.86, H 6.87, N12.10%. Found; C 51.90, H 6.26, N 12.08%.

Molecular weight: FAB-MS m/z 956 (M+Na)⁺

Infrared absorption spectrum (attached FIG. 10): ν_(max) ^(KBr) 3360,2950, 1730, 1700, 1680, 1660, 1640, 1530, 1460, 1380, 1330, 1290, 1250,1200, 1170, 1160, 1140, 1080, 980, 940, 920, 880 cm⁻¹

    ______________________________________                                        .sup.1 H Nuclear magnetic resonance spectrum (attached FIG. 11):              (400 MHz, CD.sub.3 OD) δ                                                ______________________________________                                        7.20-7.10       (3H, m)                                                       6.85-6.77       (3H, m)                                                       6.73            (1H, s)                                                       6.46            (1H, s)                                                       5.46            (1H, m)                                                       5.18            (1H, m)                                                       4.84            (1H, s)                                                       4.73            (1H, m)                                                       4.64            (1H, m)                                                       4.50            (1H, m)                                                       3.99-3.87       (2H, m)                                                       3.58-3.46       (2H, m)                                                       3.01            (1H, m)                                                       2.90            (3H, s)                                                       2.87            (1H, m)                                                       2.53            (1H, m)                                                       2.38            (1H, dd, J=14 and 11Hz)                                       2.30-2.16       (2H, m)                                                       2.00            (1H, m)                                                       1.99            (3H, s)                                                       1.84            (1H, m)                                                       1.75            (3H, d, J=7Hz)                                                1.76-1.55       (4H, m)                                                       1.43            (3H, d, J=6.5Hz)                                              1.15            (1H, m)                                                       1.00            (3H, d, J=6.5Hz)                                              0.95            (3H, d, J=6.5Hz)                                              ______________________________________                                    

    ______________________________________                                        .sup.13 C Nuclear magnetic resonance spectrum (attached FIG. 12):             (100 MHz, CD.sub.3 OD) δ                                                ______________________________________                                        176.4                (s)                                                      174.6                (s)                                                      173.4                (s)                                                      173.0                (s)                                                      172.4                (s)                                                      171.4                (s)                                                      170.4                (s)                                                      165.8                (s)                                                      160.3                (s)                                                      145.9                (s)                                                      145.9                (s)                                                      137.5                (s)                                                      134.0                (d)                                                      131.4                (s)                                                      130.6                (d) × 2                                            129.8                (s)                                                      129.1                (d) × 2                                            129.1                (s)                                                      127.6                (d)                                                      119.0                (d)                                                      118.0                (d)                                                      76.0                 (d)                                                      73.5                 (d)                                                      63.1                 (d)                                                      61.3                 (d)                                                      57.1                 (d)                                                      53.9                 (d)                                                      52.7                 (d)                                                      50.5                 (d)                                                      39.9                 (t)                                                      36.1                 (t)                                                      31.8                 (q)                                                      31.0                 (t)                                                      30.7                 (d)                                                      29.9                 (t)                                                      29.6                 (t)                                                      25.3                 (t)                                                      22.4                 (q)                                                      22.3                 (t)                                                      20.2                 (q)                                                      19.5                 (q)                                                      19.4                 (q)                                                      13.3                 (q)                                                      ______________________________________                                    

DERIVATIVES OF WS7622A SUBSTANCE

Derivatives of WS7622A substance of this invention can be represented bythe following formula (I): ##STR1## wherein R¹ and R² are each loweralkyl group or lower alkanoyl group.

A lower alkyl group means one having 1 to 6 carbon atoms, and preferredexamples of the lower alkyl group are methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tert-butyl, pentyl or the like.

A lower alkanoyl group means one having 1 to 6 carbon atoms, andpreferred examples of the lower alkanoyl are formyl, acetyl, propionyl,butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl or thelike.

Derivatives of WS7622A substance (I) can be prepared by the followingmethods:

(1) Process 1 (Alkylation of WS7622A substance): ##STR2##

Di-alkylated WS7622A substance (I^(a)) or its salt can be prepared byreacting the WS7622A substance (II) or its salt with an alkylatingagent.

Preferred examples of the salt of compounds (I^(a)) and (II) may includethe same as those exemplified as pharmaceutically acceptable salts ofWS7622A, B, C and D substances and derivatives thereof.

Preferred examples of the alkylating agent may include diazoalkanes(e.g. diazomethane, diazoethane, etc.), alkyl halides (e.g. methyliodide, ethyl iodide, etc.), dialkyl sulfates (e.g. dimethyl sulfate,etc.) and the like.

This reaction is preferably conducted in a solvent inert to thereaction, such as alcohol (e.g. methanol, ethanol, propanol, etc.),chloroform, or a mixture thereof, at ambient temperature.

In some cases, this reaction is preferably carried out in the presenceof a conventional base.

(2) Process 2 (Acylation of WS7622A substance): ##STR3##

Di-acylated WS7622A substance (I^(b)) or its salt can be prepared byreacting the WS7622A substance (II) or its salt with a compound of theformula:

    R--OH

wherein R is lower alkanoyl, or its reactive derivatives.

Preferred examples of salt of the compounds (I^(b)) and (II) may includethe same as those exemplified as pharmaceutically acceptable salts ofWS7622A, B, C and D substances and derivatives thereof.

Said reactive derivative may include acid halides, acid azides, acidanhydrides, active amides, active esters and the like.

When a free carboxylic acid (i.e. a compound of the formula: R--OH) isused, this reaction is preferably conducted in the presence of aconventional condensing agent.

This reaction is preferably conducted in a conventional solvent such asalcohol (e.g. methanol, ethanol, propanol, etc.), under ice-cooling orat ambient temperature, and good results are obtained in most cases whenthis reaction is carried out in the presence of a base such as pyridine.Such a base which is liquid may serve also as solvents.

A pharmaceutically acceptable salt of each of the WS7622A, B, C and Dsubstances and derivatives thereof may include a salt with an inorganicor organic base such as an alkali metal salt (e.g. sodium salt,potassium salt, etc.), an alkaline earth metal salt (e.g. calcium salt,etc.), ammonium salt, ethanolamine salt, triethylamine salt,dicyclohexylamine salt or the like, and an acid addition salt withorganic or inorganic acid such as methane sulfonate, hydrochloride,sulfate, nitrate, phosphate or the like.

The WS7622A, B, C and D substances, derivatives thereof and theirpharmaceutical acceptable salt have a human leukocyteelastase-inhibiting activity and is useful as human leukocyte elastaseinhibitors for treating degenerative diseases, for example, pulmonaryemphysema, atherosclerosis, rheumatoid arthritis, osteoarthritis,psoriasis, pancreatitis, adult respiratory distress syndrome and thelike.

In order to illustrate the usefulness of the WS7622A, B, C and Dsubstances, derivatives thereof and their pharmaceutically acceptablesalt, pharmacological test data thereof are shown below.

Protease Inhibition Assay

(1) Preparation of crude human leukocyte elastase:

Fifty milliliters of blood were obtained from a healthy volunteer.Leukocytes were isolated from the blood by using Mono-Poly resolvingmedium (FLOW Laboratories, Australia), and were collected bycentrifugation. The remaining erythrocytes were lysed by addingdistilled water to the precipitate, and centrifuged for 30 min at 3000rpm. To the resultant precipitate, 10 ml of 2M NaClO₄ was added andelastase was extracted. After extraction for 120 min at 0° C., thesupernatant containing elastase activity was obtained by centrifugation.After adding an equal volume of chloroform to this supernatant andshaking vigorously, a clear supernatant containing elastase activity wasseparated. Ten milliliters of the HEPES buffer were added to thesupernatant and supplied for the enzyme assay.

(2) Method:

A buffer used throughout the assay was 0.1M HEPES(N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid) containing 0.5MNaCl, pH 7.5. Twenty-five microliters of 2 mM methoxysuccinyl-(Ala)₂-Pro-Val-p-nitroanilide (100 mM of dimethyl sulfoxide solution werediluted in the buffer) and 50 μl of sample (10 μl of sample in organicsolvent was diluted 5-fold in the buffer) were mixed in wells of 96well-microtiter plate. An absorbance of the mixture in wavelength at 415nm was measured by a microplate reader (Corona Electric Co., Ibaraki,Japan). After the measurement, 25 μl of 6 μg/ml human sputum elastase(HSE) or 25 μl human leukocyte elastase preparation was added and standfor 30 min at room temperature. Then, the absorbance at 415 nm wasmeasured. Percent inhibition by drug was determined by 100×(1-"r"inhibitor present/"r" inhibitor absent), where "r" is absorbance after30 min incubation minus absorbance before enzyme addition. Effects ofinhibitors against other proteases were assayed similarly usingN-succinyl-(Ala)₃ -p-nitroanilide for porcine pancrease elastase (TypeIV, 5 μg/ml final), N-alpha-benzoyl-Arg-p-nitroanilide for bovinepancrea trypsin (Type I, 16 μg/ml final), methoxysuccinyl-(Ala)₂-Pro-Met-p-nitroanilide for bovine pancreas chymotrypsin (Type II, 1.5μg/ml final) and for human sputum cathepsin G (10 unit/ml final). HSEand cathepsin G were obtained from Elastin Products Company Inc., MO.,U.S.A. All other substrates and proteases were purchased from SigmaChemicals Co.

(3) Result:

    __________________________________________________________________________    1) Inhibitory effect of WS7622A, B, C and D                                   substances on several serine protease activity                                              Porcine   Chymo-                                                                             Human                                            Substance                                                                           Human sputum                                                                          pancreas                                                                           Trypsin                                                                            trypsin                                                                            leukocyte                                                                          Cathep-                                     (μg/ml)                                                                          elastase                                                                              elastase                                                                           (bovine)                                                                           (bovine)                                                                           elastase                                                                           sin G                                       __________________________________________________________________________    WS7622A                                                                             0.0071  <0.005                                                                             >250 0.031                                                                              0.017                                                                              1.1                                         WS7622B                                                                             0.038    0.013                                                                             >250 0.13 0.029                                                                              1.85                                        WS7622C                                                                             0.12     0.036                                                                             >5   0.21                                                  WS7622D                                                                             0.15     0.12                                                                              >5   0.38                                                  __________________________________________________________________________

    ______________________________________                                        2) Inhibitory effect of derivatives of WS7622A                                substance on human leukocyte elastase                                                       μg/ml                                                        ______________________________________                                        The compound    0.0103                                                        of Example 2                                                                  The compound    0.0103                                                        of Example 3                                                                  ______________________________________                                    

Each value was expressed as 50% inhibitory concentration.

Pharmaceutical compositions of this invention can be used in aconventional pharmaceutical forms such as powders, fine granules,granules, tablets, dragee, microcapsules, capsules, suppository,solution, suspension, emulsion, syrups and the like. If desired,diluents or disintegrators (e.g. sucrose, lactose, starch, crystallinecellulose, low-substituted hydroxypropyl cellulose, synthetic aluminumsilicate, etc.), binding agents (e.g. cellulose, methylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose,polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic,polyethyleneglycol, etc.), coloring agents, sweeting agents, lubricant(e.g. magnesium stearate, etc.) or the like, may be dispensed with saidcomposition.

The dosage of said composition of this invention depends on thepatient's age, body weight, condition, etc., and it is generallyadministered by the oral route at the daily dose level of 100 mg to 10 gas the object compound or its pharmaceutically acceptable salt,preferably 1 g to 5 g on the same basis, at the interval of 1 to 3 timesa day. Typical unit doses may be 50 mg, 100 mg, 200 mg, 500 mg, 1 g andthe like, although these are only examples and not limitative, ofcourse.

The following Examples are given for the purpose of illustrating thisinvention.

EXAMPLE 1 Production of WS7622A Substance

An aqueous seed medium (200 ml) containing 1% of soluble starch, 1% ofsucrose, 1% of glucose, 1% of pharma media (cotton seed flour, tradename), 0.5% of polypeptone, 0.5% of soybean meal and 0.1% of CaCO₃ waspoured into each of twelve 500-ml Elrenmyer flasks, and sterilized at120° C. for 30 min. A loopful of Streptomyces resistomycificus No. 7622on mature slant culture was inoculated to each of the seed medium. Theflasks were shaken on a rotary shaker at 30° C. for 3 days. Theresultant seed culture was inoculated to 1601 of sterile fermentationmedium consist of 4% Pine-Dex (starch acid hydrolysate, trade name), 1%gluten meal, 0.5% wheat germ, 0.5% potato protein and 0.2% CaCO₃ in a2001 stainless steel jar-fermentor. The fermentation was carried out at25° C. for 5 days under aeration of 1601/min and agitation of 200 rpm.

An amount of the WS7622A substance in the fermentation broth wasquantified by elastase inhibition assay in vitro. The sample for theassay was prepared as follows;

An equal volume of acetone was added to a broth with vigorous stirring.The mixture was allowed to stand at room temperature for 1 hour and thenfiltered. The filtrate was concentrated under reduced pressure to anappropriate volume. The elastase inhibition assay was described before.

The cultured broth (1601) was filtered with the aid of diatomaseousearth. Fifty liter of acetone was added to the mycelial cake withstirring. The mixture was allowed to stand at room temperatureovernight, and then filtered. The filtrate was concentrated to removethe acetone under reduced pressure. The filtrate (1401) from the brothobtained in the above and the mycelial extract were combined, and thenpassed through a column of polymeric adsorbent, Diaion HP-20 (tradename, made by Mitsubishi Chemical Industrial Limited, 171). The columnwas washed with 501 of water and 50% of aqueous methanol solution (501),and the adsorbate was eluted with 401 of methanol. The eluate wasconcentrated under reduced pressure to give an oily residue. The residuewas applied to a column chromatography on silica gel (Kiesel gel 60,70-230 mesh, Merck, 1.31). The column was washed with 21 ofn-hexane-ethyl acetate (1:1, V/V) and 41 of ethyl acetate and the activesubstance was eluted from the column with acetone (31) andacetone-methanol (10:1, 61). The active fractions (61) were combined andconcentrated to dryness, and was subjected to a column chromatography onsilica gel with stepwised elution using solvents of chloroform-methanolmixture. The active substance was eluted in chloroform-methanol (10:1,V/V) solution. The fractions were concentrated and dried under reducedpressure to give 3 g of yellow powder. WS7622A substance was separatedby High Performance Liquid Chromatography (HPLC). A YMC-D-ODS-15B 30×250mm stainless steel column (Yamamura Chemical Laboratories, Japan) packedwith s-15 reverse phase silica was used. Fifty milligram of the yellowpowder was dissolved in 50 μl of methanol and applied to the HPLC with60% of aqueous methanol solution as mobile phase and flow rate of 20ml/min. The retention time of WS7622A substance was 17.6 min. Thechromatogram was run 60 times, and the fractions containing WS7622Asubstance were combined and concentrated dryness. The residue wasdissolved with small amount of methanol and allowed to stand over nightto give 600 mg of WS7622A substance as colorless prism.

EXAMPLE 2 Production of Dimethylated WS7622A Substance R¹, R² : methyl

To a solution of WS7622A substance (1 g) in a mixture of chloroform (20ml) and methanol (20 ml) was added trimethylsilyldiazomethane (4 ml, 10%weight in hexane, purchased from Petrarch Systems Co., Ltd.) and thesolution was allowed to stand at room temperature overnight. Thesolution was evaporated to dryness to give an oil which was purified bycolumn chromatography on silica gel eluting with a mixture of chloroformand methanol (95:5). The product fractions were collected and evaporatedto dryness. The obtained white residue was triturated with diethyl etherto give 0.62 g of dimethylated WS7622A substance as white powder.

Appearance: white powder

Molecular formula: C₄₉ H₆₇ N₉ O₁₃

FAB-MS m/z: 990 (M+H)⁺

Thin Layer Chromatography (TLC)

Silica gel plate

    ______________________________________                                        (Merck Art 5715)                                                                             chloroform-methanol                                                                          Rf                                                             (5:1, V/V)     0.72                                                           chloroform-methanol                                                                          Rf                                                             (9:1, V/V)     0.40                                            ______________________________________                                    

Specific rotation: [α]_(D) ²³ +37° (C=1.6, CHCl₃ -MeOH (1:1))

Infrared absorption spectrum: ν_(max) ^(Nujol) 3400, 3250, 1730, 1660,1640, 1540, 1520, 1330, 1260, 1210, 1180, 1160, 1100, 1080, 1000, 980,920 cm⁻¹

    ______________________________________                                        .sup.1 H Nuclear magnetic resonance spectrum:                                 (400 MHz, CD.sub.3 OD) δ                                                ______________________________________                                        7.20-7.11          (3H, m)                                                    6.93               (1H, s)                                                    6.83               (1H, q, J=7Hz)                                             6.77               (2H, d, J=8Hz)                                             6.59               (1H, s)                                                    5.48               (1H, m)                                                    5.15               (1H, m)                                                    4.85               (1H, s)                                                    4.76               (1H, m)                                                    4.67               (1H, m)                                                    4.42               (1H, m)                                                    3.99-3.89          (2H, m)                                                    3.82               (3H, s)                                                    3.81               (3H, s)                                                    3.66               (1H, m)                                                    3.55               (1H, m)                                                    3.02               (1H, m)                                                    2.95               (3H, s)                                                    2.90               (1H, m)                                                    2.60-1.94          (6H, m)                                                    1.82               (1H, m)                                                    1.77               (3H, d, J=7Hz)                                             1.68-1.58          (4H, m)                                                    1.46               (3H, d, J=7Hz)                                             1.13               (3H, d, J=6Hz)                                             1.12               (3H, d, J=6Hz)                                             1.01               (3H, d, J=6Hz)                                             0.96               (3H, d, J=6Hz)                                             1.00               (1H, m)                                                    ______________________________________                                    

EXAMPLE 3 Production of Diacetylated WS7622A Substance (R¹, R² : acetyl

To a solution of WS7622A substance (1 g) in pyridine (5 ml) was addedacetic anhydride (0.24 ml) and the mixture was allowed to stand at roomtemperature overnight. The mixture was evaporated to dryness to give anoil which was purified by column chromatography on silica gel elutingwith a mixture of chloroform and methanol (95:5). Crystallization fromhot methanol-isopropyl ether gave 0.73 g of diacetylated WS7622Asubstance as a white powder.

Appearance: colorless prism

Molecular formula: C₅₁ H₆₇ N₉ O₁₅

FAB-MS m/z: 1046 (M+H)⁺

Thin layer chromatography:

Silica gel plate

    ______________________________________                                        (Merck Art 5715)                                                                             chloroform-methanol                                                                          Rf                                                             (5:1, V/V)     0.73                                                           chloroform-methanol                                                                          Rf                                                             (9:1, V/V)     0.40                                            ______________________________________                                    

Specific rotation: [α]_(D) ²³ +29° (C=1.0, CHCl₃ -MeOH (1:1))

Infrared absorption spectrum: ν_(max) ^(Nujol) 3400, 1770, 1730, 1660,1640, 1540, 1420, 1340, 1260, 1210, 1080, 1010, 980, 920 cm⁻¹

    ______________________________________                                        .sup.1 H Nuclear magnetic resonance spectrum:                                 (400 MHz, DMSO-d.sub.6) δ                                               ______________________________________                                        9.48            (1H, broad signal)                                            8.37            (1H, d, J=8Hz)                                                7.76            (1H, d, J=7Hz)                                                7.23-7.10       (4H, m)                                                       7.18            (1H, s)                                                       7.03            (1H, d, J=9Hz)                                                6.93            (1H, s)                                                       6.76            (2H, d, J=8Hz)                                                6.74            (1H, br s)                                                    6.65            (1H, q, J=7Hz)                                                5.74            (1H, m)                                                       5.42            (1H, m)                                                       5.08            (1H, m)                                                       4.77            (1H, d, J=10Hz)                                               4.64            (1H, m)                                                       4.57            (1H, m)                                                       4.42            (1H, m)                                                       3.92-3.78       (2H, m)                                                       3.49            (1H, m)                                                       3.34            (1H, m)                                                       2.90            (1H, dd, J=13 and 12Hz)                                       2.80            (3H, s)                                                       2.70            (1H, m)                                                       2.60            (1H, dd, J=13 and 11Hz)                                       2.54            (1H, septet, J=6Hz)                                           2.42-2.27       (2H, m)                                                       2.27            (3H, s)                                                       2.25            (3H, s)                                                       2.17            (1H, m)                                                       1.88            (1H, m)                                                       1.70            (1H, m)                                                       1.66            (3H, d, J=7Hz)                                                1.60-1.46       (4H, m)                                                       1.34            (3H, d, J=6Hz)                                                0.99            (6H, d, J=6Hz)                                                0.91            (3H, d, J=6Hz)                                                0.88            (3H, d, J=6Hz)                                                0.90            (1H, m)                                                       ______________________________________                                    

EXAMPLE 4 Production of WS7622B Substance

An aqueous seed medium (200 ml) containing 1% of soluble starch, 1% ofsucrose, 1% of glucose, 1% of pharma media (cotton seed fluor, tradename), 0.5% of polypeptone, 0.5% of soybean meal and 0.1% of CaCO₃ waspoured into each of twelve 500-ml Elrenmyer flasks, and sterilized at120° C. for 30 min. A loopful of Streptomyces resistomycificus No. 7622of mature slant culture was inoculated to each of the seed medium. Theflasks were shaken on a rotary shaker at 30° C. for 3 days. Theresultant seed culture was inoculated to 1601 of sterile fermentationmedium consist of 4% Pine-Dex {starch acid hydrolyste trade name), 1%gluten meal, 0.5% wheat germ, 0.5% potato protein and 0.2% CaCO₃ in a2001 stainless steel jar-fermentor. The fermentation was carried out at25° C. for 5 days under aeration of 1601/min and agitation of 200 rpm.

An amount of the WS7622B substance in the fermentation broth wasquantified by elastase inhibition assay in vitro. The sample for theassay was prepared as follows;

An equal volume of acetone was added to a broth with vigorous stirring.The mixture was allowed to stand at room temperature for 1 hour and thenfiltered. The filtrate was concentrated under reduced pressure to anappropriate volume. The elastase inhibition assay was described before.

The cultured broth (1601) was filtered with the aid of diatomaseousearth. Fifty liter of acetone was added to the mycelial cake withstirring. The mixture was allowed to stand at room temperatureovernight, and then filtered. The filtrate was concentrated to removethe acetone under reduced pressure. The filtrate (1401) from the brothobtained in the above and the mycelial extract were combined, and thenpassed through a column of polymeric adsorbent, Diaion HP-20 (tradename, made by Mitsubishi Chemical Industrial Limited, 171). The columnwas washed with 501 of water and 50% of aqueous methanol solution (501),and the adsorbate was eluted with 401 of methanol. The eluate wasconcentrated under reduced pressure to give an oily residue. The residuewas applied to a column chromatography on silica gel (Kiesel gel 60,70-230 mesh, Merck, 1.31). The column was washed with 21 ofn-hexane-ethyl acetate (1:1, V/V) and 41 of ethyl acetate and the activesubstance was eluted from the column with acetone (31) andacetone-methanol (10:1, 61). The active fractions (61) were combined andconcentrated to dryness, and was subjected to a column chromatography onsilica gel with stepwised elution using solvents of chloroform-methanolmixture. The active substance was eluted with chloroform-methanol(10:1). The fractions were concentrated and dried under reduced pressureto give 3 g of yellow powder. WS7622B substance was separated by HighPerformance Liquid Chromatography (HPLC). A YMC-D-ODS-15B 30×250 mmstainless steel column (Yamamura Chemical Laboratories, Japan) packedwith s-15 reverse phase silica was used. Fifty milligram of the yellowpowder was dissolved in 50 μl of methanol and applied to the HPLC with60% of aqueous methanol solution as mobile phase and flow rate of 20ml/min. The retention time of WS7622B substance was 23.0 min. Thechromatogram was run 60 times, and the fractions containing WS7622Bsubstance were combined and concentrated, and then crystallized withmethanol to give 180 mg of WS7622B substance as colorless needles.

EXAMPLE 5 Production of WS7622C and D Substances

The fermentation was carried out in the same manner as that of Example1.

To the cultured broth (70 L) thus obtained, seventy liters of methanolwere added with stirring. The mixture was allowed to stand at roomtemperature for 1 hour, and then filtered. One hundred and forty litersof water was added to the filtrate and 2801 of 25% aqueous methanolsolution was made. The solution was passed through a column of polymericadsorbent, Diaion HP-20 (trade name, made by Mitsubishi ChemicalIndustries Limited, 51). The column was washed with 101 of 50% aqueousmethanol solution and eluted with 101 of methanol. The eluate wasconcentrated under reduced pressure to give an oily residue. The residuewas applied to a column chromatography on silica gel (Kiesel gel 60,70-230 mesh, Merck, 2.21). The column was washed with 101 of acetone andthe active substances were eluted from the column with acetone-methanol(5:1). Fraction collection of the eluate was as follows; fraction No. 1:11, fraction No. 2: 5.41, fraction No. 3: 3.81, fraction No. 4: 3.21.WS7622C and D substances were eluted at fraction No. 3 and fraction No.4, respectively.

Isolation of WS7622C Substance

Fraction No. 3 was concentrated and dried under reduced pressure to give1.2 g of a yellow powder. One gram of the powder containing WS7622Csubstance was redissolved in 6 ml of chloroform-methanol (10:1) andsubjected to 100 ml of silica gel column, prepacked withchloroform-methanol (10:1). The column was washed with 300 ml ofchloroform-methanol (10:1) and WS7622C substance was eluted withchloroform-methanol (5:1). The first 180 ml of the eluate was discardedand subsequent 80 ml were pooled and concentrated and dried underreduced pressure to give 372 mg of powder. The powder was redissolved in6 ml of ethanol and stand at 4° C. for 1 hour gave 170 mg of WS7622Csubstance as colorless needles.

Isolation of WS7622D Substance

Fraction No. 4 was concentrated to dryness gave 3.3 g of a yellowpowder. One gram of the powder containing WS7622D substance wasdissolved in 6 ml of chloroform-methanol (20:1) and subjected to 100 mlof silica gel column, prepacked with chloroform. The column was washedwith 300 ml of chloroform-methanol (20:1) and 300 ml ofchloroform-methanol (10:1) and WS7622D substance was eluted withchloroform-methanol (5:1). The first 200 ml of the eluate was discardedand subsequent 90 ml were pooled and concentrated to dryness gave 127 mgof powder. The powder was dissolved in a small amount of ethanol andstand at 4° C. overnight gave 90 mg of WS7622D substance as colorlessneedles.

We claim:
 1. A method of treating pulmonary emphysema or adultrespiratory distress syndrome in a subject in need thereof whichcomprises administering to the subject an effective amount of WS7622A,B, C and/or D, or di-lower alkyl derivative of WS7622A substance, ordi-lower alkanoyl derivative of WS7622A substance, or theirpharmaceutically acceptable salt.